1. Technical Field
The present invention relates to the field of plasmonic sensors and optical sensors, in particular to integrated spectroscopic plasmonic sensor devices, integrated plasmonic biosensor devices for oligonucleotide and protein analysis, and integrated plasmonic sensors, in particular, integrated plasmonic micro-array devices for nucleic acid and protein analysis.
2. Description of the Related Art
Spectrometers are devices used to measure properties of light over a portion of the electromagnetic spectrum. The properties of light measured include the intensity of the light and the polarization state of the light. The independent variable is often the wavelength of light or a measure of the photon energy of the light. Spectrometers usually employ a source of electromagnetic energy, various optical devices such as mirrors and gratings as optical interference filters for dispersing the light to the detector, and a detector to detect the light intensity or photon energy as a function of wavelength. Existing devices for detecting the light properties include electronic photodetectors such as charge-coupled devices (CCD), CMOS active pixel sensor arrays, or focal plane arrays. Existing spectrometers find use in many areas, including analyzing compositions of unknown material.
Micro-array technology for expression profiling is well known and is currently widely used in genomic research. Recently much interest has centered on the development of DNA chips based on high density oligonucleotide arrays and fluorescence analysis such as described by Hacia et al. (G. Hacia, L. C. Brody, M. S. Chee. S. P. A. Fodor F. S. Collins in Nature Genetics 14, December 1996). One of the examples of commercialization of this technology has been Affymetrix's “GeneChip”, which was developed to process large amounts of genetic information. Affymetrix technology relies on photolithographic processing to produce thousands of detection regions on a single chip. Alternative techniques include robotic spotting and ink-jet printing although they achieve somewhat smaller detection region density within the micro-array.
For the micro-arrays in common use, one typically starts by taking a specific biological substance or system of interest, extracting its mRNA, and making a fluorescence tagged cDNA copy of this mRNA. This tagged cDNA copy, typically called the target, is then hybridized to a slide containing a grid or array of single stranded DNA (ssDNA) called probes which have been built or placed (i.e. immobilized) in specific detection regions on this grid. Similar to the general hybridization principle, a target will only hybridize with its complementary probe i.e., nucleic acid strands tend to be paired to their complements in double-stranded structures. Thus, a single-stranded cDNA target molecule will seek out its complement in a complex mixture of ssDNA containing large numbers of other nucleic acid molecules. Hence, nucleic acid probe (e.g., gene probe) detection methods are very specific to DNA sequences. Factors affecting the hybridization or re-association of two complementary DNA strands include temperature, contact time, salt concentration, the degree of mismatch between the base pair sequences, and the length and concentration of the target and probe sequences. In perhaps the simplest procedure, hybridization is performed on an immobilized probe molecule attached on a solid surface such as a nitrocellulose or nylon membrane or a glass plate.
Fluorescent markers are typically added to the target in one of two ways: (i) fluorescent nucleotide bases are used when making the cDNA copy of the mRNA or (ii) biotinylated nucleotides are first incorporated, followed by an application of fluorescent marker-labeled streptavidin, which will bind to biotin (S. Kohane “Microarrays for integrated genomics” MIT Press, 2002).
Depending on manufacturer specific protocols, the target-probe hybridization process on a microarray typically occurs over several hours. All unhybridized targets are then washed off and the microarray is illuminated with laser light and scanned using laser confocal microscopy. A digital image scanner records the brightness level at each grid location on the micro-array corresponding to particular probe species. The brightness level is correlated with the absolute amount of mRNA in the original sample, and by extension, the expression level of the gene associated with this mRNA.
DNA and protein micro-array technology has yet to be successfully developed into monolithically integrated single chip devices that conveniently and inexpensively capture, deliver and interpret information. What is currently understood by the term “biochip” is typically a glass slide with an array of detection regions, each region containing specific probe molecules, which requires complex and bulky equipment for external laser excitation, scanning and imaging of the optical signals. In addition to the cost associated with this equipment, there is also a requirement for it to be operated by highly trained and skilled personnel in order to ensure error free interpretation of the gathered data and troubleshooting. These limitations of cost and space associated with the present status of biochip technology currently prevent DNA and protein analysis from finding a wider use in hospital, point-of-care, and limited resource settings.
Over the past few years there has been some effort deployed to reduce the cost and size of biochips by integrating them with the associated laser excitation and image scanning apparatus (Vo-Dinh et al., “Integrated circuit biochip microsystem” U.S. Pat. No. 6,448,064, September 2002; Duvenecket et al., “Optical detection device based on semiconductor laser array” U.S. Pat. No. 6,469,785, October 2002; Bruno-Raimondi et al “Sensing unit provided with separated detection light guiding” U.S. Pat. No. 6,437,345, August 2002; Neuschafer et al “Sensor platform and method for the parallel detection of a plurality of analytes using evanescently excited luminescence” U.S. Pat. No. 6,078,705, June 2000). These documents proposed an integrated circuit biochip micro system, which combines lasers, detectors, focusing optics and biological sensing elements within a single micro-assembly. In microelectronics this type of integration is typically defined as hybrid integration, i.e., when individual elements are produced separately by processing a number of separate substrates/wafers and then diced out and microassembled together. Although advantageous over the bulky, bench-top devices, such hybrid integrated biochips still lack the cost and performance advantage of true monolithic integration. In addition, these devices require the use of fluorescent markers which unnecessarily complicates the analysis procedure and which ideally should be avoided in a simple point-of-care device.
An optoelectronic biochip sensor has previously been proposed using light sources for determining a molecular recognition event on the basis of monolithically integrated dielectric waveguide components (Bazylenko et al., “Integrated circuit biochip microsystem” U.S. Pat. No. 7,768,650 issued Aug. 3, 2010). While such sensors will typically meet sensitivity requirements for useful sensors, their dimensions are limited by diffraction effects in the dielectric waveguide components used. Such sensors can therefore be less attractive for integration with silicon based electronic components, such as complementary metal oxide semiconductor (CMOS) circuits, which are many times smaller.
Excitations which do not exhibit the disadvantages associated with using light sources to determine a specific binding event are so-called surface plasmons polaritons or plasmonic mode excitations, i.e., electromagnetic excitations at a metal-dielectric interface. These excitations may be guided using structures that are much smaller than the wavelength of photons of the same frequency. Discrete devices for generating such plasmons are known for example from the article by Walters et al., “A silicon-based electrical source of surface plasmon polaritons”, Nature Materials, 6 Dec. 2009. Such plasmonic mode excitations provide an opportunity to develop methods and devices configured to utilize plasmonic sources to determine molecular recognition events.
Surface plasmon resonance (SPR) measurement systems can be used to detect shifts in refractive indices of samples associated with SPR sensors that are included in the systems. A conventional SPR measurement system includes an external light source that illuminates an SPR sensor through an input optical path that typically includes a prism. Light reflected by the SPR sensor propagates through an output optical path and is intercepted by a detector. The optical paths typically include telescopes, polarizers, acousto-optic deflectors, and other optical components that add complexity to the SPR measurement systems, especially when the SPR sensor includes an array of sensing elements. To accommodate an array of sensing elements, the output optical path includes an imaging system that maps each sensing element in the array to a corresponding detection element within the detector. The imaging system can increase the manufacturing cost of the SPR measurement system and can limit the physical density of the sensing elements in the array due to the limited registration that can be achieved by a typical imaging system. An opto-electric SPR biosensor that is illuminating from below through a supporting substrate wafer is known from Bahl et al., “Integrated Opto-electric SPR Sensor”, US 2007/0052049 A1, Mar. 8, 2007. The biosensor of Bahl et al. suffers from fragility due to the prismatic coupling structures that are etched into the substrate and limited integration density due to the ray optics principles that guide the design.
A further known limitation of the conventional SPR technique is its relatively low sensitivity, which is typically between 10−3 and 10−5 refractive index units (RIU) although the sensitivity can, in some circumstances, be improved up to 10−6 RIU. However, for modern demanding bio-chemical applications, a sensitivity of about 10−9 RIU or better is essential. Thus, a more advanced SPR technique has been applied in bio-chemical sensors. This more advanced SPR technique is based on the application of the Goos-Hanchen (GH) effect. In some sensors, the GH effect is small and not useful for sensing measurements. In other sensors, the GH effect is more substantial and is used to improve evanescent-wave propagation. Advanced biosensors exploiting GH effect phenomena associated with plasmonic mode excitations are known for example from Potyrailo et al., “Methods and systems for detecting biological and chemical materials on a submicron structured substrate”, US 2008/0280374 A1, Nov. 13, 2008. However these proposed biosensors are not monolithically integrated and thus require bulky and expensive external equipment for the scanning and imaging of optical signals generated by the fluorescent markers.